In pyrosequencing, a sequencing primer is hybridized to a single stranded DNA template to form a nucleic acid duplex. The duplex is incubated with the enzymes DNA polymerase, ATP sulfurylase, and luciferase, and with the substrates adenosine 5′-phosphosulfate (APS) and luciferin. In addition, one of the four deoxynucleotide triphosphates (dNTPs; i.e., dATP, dGTP, dGTP, or TTP) is added. If the dNTP is complementary to the next position on the nucleic acid template, the deoxynucleotide is incorporated into the elongating nucleic acid strand, and a pyrophosphate (PPi) molecule is released stoichiometrically. Importantly, pyrophosphate is only released if the complementary nucleotide is incorporated into the elongating nucleic acid chain.
The release of PPi indicates that a reaction has occurred. Consequently, the identity of the nucleotide incorporated into the elongating nucleic acid strand can be determined based on whether or not PPi has been released. The pyrophosphate released in these reactions can be detected in a variety of ways, including enzymatically. First, sulfurylase quantitatively converts PPi to adenosine triphosphate (ATP) in the presence of APS. Subsequently, this ATP is consumed in a luciferin-luciferase reaction to generate PPi and detectable light in amounts that are proportional to the amount of ATP. Thus, each light signal is proportional to the number of nucleotides incorporated into the growing strand. Moreover, pyrosequencing depends upon the release of PPi to initiate the cascade of reactions resulting in the emission of a detectable light signal. Such methods allow a base to be identified at a target position. Successive iterations of the method with each dNTP allow a DNA template to be sequenced.